Biology subjects

Khadayate, S.

Publications and source records attributed to Khadayate, S..

6 recordsLinked to original sources

bioRxiv2026

Mechanisms of MCM2-7 helicase activation and initial DNA melting at near base-pair resolution

During eukaryotic DNA replication initiation, inactive MCM2-7 double-hexamers assembled at replication origins must be converted into two active CMG helicases, yet how this transition is coupled to origin DNA unwinding in vivo remains unclear. Here, we identify a DNA-bound intermediate with an extended genomic footprint that forms during helicase activation. Genome-wide mapping of initial strand separation reveals that DNA unwinding initiates near the N-terminal interface of opposing MCM2-7 hexamers. At these sites, the origin DNA exhibits a conserved AT-rich/GC-rich/AT-rich sequence architecture on which the helicase complex is centred, consistent with a role in promoting DNA extrusion following double-hexamer separation. We further show that restricting hexamer rotation and splitting delays release of the Cdc45-loading factor Sld3, demonstrating that mechanical transitions during helicase activation are tightly coupled to complex disassembly. Finally, we provide in vivo evidence that single-stranded DNA is ejected through a specialised DNA exit gate at the Mcm2/5 interface during helicase activation, which is dispensable for ongoing DNA synthesis. Together, these findings establish a mechanistic framework for how replication origins are remodelled to initiate DNA replication and reveal key intermediates and DNA transactions during helicase activation.

molecular biology↗

bioRxiv2023

Ribosomal S6 kinase 1 regulates inflammaging via the senescence secretome

Inhibition of the nutrient-responsive mTOR (mammalian target of rapamycin) signalling pathway including the key downstream effector S6 kinase 1 (S6K1) extends lifespan and improves healthspan in mice. However, the underlying mechanisms contributing to the broad range of age-related benefits observed with loss of S6K1 signalling are unclear. Cellular senescence is a stable growth arrest accompanied by an inflammatory phenotype (termed the senescence-associated secretory phenotype, or SASP). While both cellular senescence and SASP-mediated chronic inflammation contribute to age-related pathology, the specific role of S6K1 signalling in these processes has not been determined. Here, focussing on mouse liver, a key target tissue for the beneficial metabolic effects of loss of S6K1 signalling, we show that S6K1 deletion does not reduce senescence but ameliorates inflammation and immune cell infiltration in aged livers. Using human and mouse models of senescence, we demonstrated that reduced inflammation is a liver-intrinsic effect associated with S6K deletion. Furthermore, gene expression analysis suggested that downregulated cGAS/STING and IRF3 activation might mediate the impaired SASP observed upon S6K deletion. Using a hepatic oncogene induced senescence model, we showed in vivo that S6K1 deletion results in reduced IRF3 activation, impaired production of cytokines such as IL1y and reduced immune infiltration. Overall, deletion of S6K reduces inflammation in the liver suggesting that suppression of the inflammatory SASP by loss of S6K could contribute to explain the beneficial effects of inhibiting this pathway on healthspan and lifespan.

physiology↗

bioRxiv2023

Metabolomic and transcriptomic analyses of Fmo5-/- mice reveal roles for flavin-containing monooxygenase 5 (FMO5) in NRF2-mediated oxidative stress, the unfolded protein response, lipid homeostasis, and carbohydrate and one-carbon metabolism

Flavin-containing monooxygenase 5 (FMO5) is a member of the FMO family of proteins best known for their roles in the detoxification of foreign chemicals and more recently in endogenous metabolism. We have previously shown that Fmo5-/- mice display an age-related lean phenotype, with much reduced weight gain from 20 weeks of age. The phenotype is characterized by decreased fat deposition, lower plasma concentrations of glucose and cholesterol, higher glucose tolerance and insulin sensitivity, and resistance to diet-induced obesity. In the present study we report the use of metabolomic and transcriptomic analyses of livers of Fmo5-/- and wild-type mice to identify factors underlying the lean phenotype of Fmo5-/- mice and gain insights into the function of FMO5. Disruption of the Fmo5 gene has wide-ranging effects on the abundance of metabolites and expression of genes in the liver. The results reveal that FMO5 is involved in upregulating the NRF2-mediated oxidative stress response, the unfolded protein response and response to hypoxia and cellular stress, indicating a role for the enzyme in adaptation to oxidative and metabolic stress. FMO5 also plays a role in stimulating a wide range of metabolic pathways and processes, particularly ones involved in the regulation of lipid homeostasis, the uptake and metabolism of glucose, the generation of cytosolic NADPH, and in one-carbon metabolism. The results predict that FMO5 acts by stimulating the NRF2, XBP1, PPARA and PPARG regulatory pathways, while inhibiting STAT1 and IRF7 pathways.

biochemistry↗

bioRxiv2023

Establishment of beta cell heterogeneity via differential CpG methylation at Nnat

Aims/hypothesisBeta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly-connected hub cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility which we explore here by focussing on the imprinted gene neuronatin (Nnat), which is required for normal insulin synthesis and secretion. MethodsSingle cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing eGFP under the control of the Nnat enhancer/promoter regions were generated for fluorescence-activated cell (FAC) sorting of beta cells and downstream analysis of CpG methylation by bisulphite and RNA sequencing, respectively. Animals deleted for the de novo methyltransferase, DNMT3A from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 and Cal-590. Insulin secretion was measured using Homogeneous Time Resolved Fluorescence Imaging. ResultsNnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic data sets demonstrated the early establishment of Nnat-positive and negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a sub-population specialised for insulin production, reminiscent of recently-described "{beta}HI" cells and were diminished in db/db mice. Hub cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialization. Conclusions/interpretationThese findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may thus contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. Research in contextWhat is already known about this subject? - Neuronatin (Nnat/NNAT) is an imprinted gene in humans and mice and is required for glucose-stimulated insulin secretion in vivo - Pancreatic beta cells are functionally heterogeneous with specific highly-connected subpopulations known to coordinate islet wide Ca2+ dynamics - Functional subpopulations have been described in human beta cells and their distribution is altered in type 2 diabetes What is the key question? - Does NNAT mark a discrete subpopulation of functional beta cells and which epigenetic pathways coordinate its formation and maintenance? What are the new findings? - A subpopulation of NNAT+ beta cells is established prior to the first week of postnatal life in mice via de novo DNA methylation at the Nnat promoter - NNAT+ beta cells are transcriptionally highly differentiated and appear to be functionally specialised for insulin production, possibly corresponding to recently-described "{beta}HI" and "CD63hi" beta cells. NNAT is expressed in a subset of beta cells across the human islet, and its deficiency in human beta cells diminishes glucose-stimulated insulin secretion - NNAT+ cells are likelier to belong to the population of follower, rather than hub cells, consistent with a role in insulin production rather than glucose detection How might this impact on clinical practice in the foreseeable future? - Epigenome-modifying compounds may provide a way of enhancing beta cell function and the ensemble behaviour of the islet to stimulate insulin secretion

cell biology↗

bioRxiv2020

RUNX1 controls the dynamics of cell cycle entry of naive resting B cells by regulating expression of cell cycle and immunomodulatory genes in response to BCR stimulation

RUNX1 is a transcription factor that plays key roles in haematopoietic development and in adult haematopoiesis and lymphopoiesis. Here we report that RUNX1 is also involved in controlling the dynamics of cell cycle entry of naive resting B cells in response to stimulation of the B cell receptor (BCR). Conditional knockout of Runx1 in mouse resting B cells resulted in accelerated entry of the cells into S-phase following BCR engagement. Our results indicate that Runx1 regulates the cyclin D2 (Ccnd2) gene, the immediate early genes, Fosl2, Atf3 and Egr2, and the Notch effector Rbpj, in B cells, reducing the rate at which transcription of these genes increases following BCR stimulation. RUNX1 interacts with the chromatin remodeller SRCAP, recruiting it to promoter and enhancer regions of the Ccnd2 gene. BCR-mediated activation triggers switching between binding of RUNX1 and its paralog RUNX3 and between SRCAP and the SWI/SNF remodelling complex member BRG1. We also find that RUNX1 regulates expression of a number of immunomodulatory genes in resting B cells. These include the interferon receptor subunit gene Ifnar1, which is upregulated in B cells from lupus patients, the Ptpn22 gene, which has been identified as a major lupus risk allele, and the Lrrk2 gene, which is mutated in familial Parkinsons disease. The hyperresponsiveness of the Runx1 knockout B cells to antigen stimulation and its role in regulating a suite of genes that are known to be associated with autoimmune disease suggest that RUNX1 is a major regulator of B cell tolerance and autoimmunity.

immunology↗

bioRxiv2019

Mesocorticolimbic circuit mechanisms underlying the effects of ketamine on dopamine: a translational imaging study

Patients with schizophrenia show increased striatal dopamine synthesis capacity in imaging studies. However, the mechanism underlying this is unclear but may be due to N-methyl-D-aspartate receptor (NMDAR) hypofunction and parvalbumin (PV) neuronal dysfunction leading to disinhibition of mesostriatal dopamine neurons. Here, we test this in a translational mouse imaging study using a ketamine model. Mice were treated with sub-chronic ketamine (30mg/kg) or saline followed by in-vivo positron emission tomography of striatal dopamine synthesis capacity, analogous to measures used in patients. Locomotor activity was measured using the open field test. In-vivo cell-type-specific chemogenetic approaches and pharmacological interventions were used to manipulate neuronal excitability. Immunohistochemistry and RNA sequencing were used to investigate molecular mechanisms. Sub-chronic ketamine increased striatal dopamine synthesis capacity (Cohens d=2.5, P<0.001) and locomotor activity. These effects were countered by inhibition of midbrain dopamine neurons, and by activation of cortical and ventral subiculum PV interneurons. Sub-chronic ketamine reduced PV expression in these neurons. Pharmacological intervention with SEP-363856, a novel psychotropic agent with agonism at trace amine receptor 1 (TAAR1), significantly reduced the ketamine-induced increase in dopamine synthesis capacity. These results show that sub-chronic ketamine treatment in mice mimics the dopaminergic alterations in patients with psychosis, and suggest an underlying neurocircuit involving PV interneuron hypofunction in frontal cortex and hippocampus as well as activation of midbrain dopamine neurons. A novel TAAR1 agonist reversed the dopaminergic alterations suggesting a therapeutic mechanism for targeting presynaptic dopamine dysfunction in patients.

neuroscience↗